The present invention relates to a method for genotyping microsatellite DNA markers using ligation of at least three oligonucleotides. Specifically, the present invention provides a method for distinguishing allele content in mono-, di-, tri-, tetra-, penta-, hexa-, hepta-, octa- or nona-repeated DNA using combinations of at least three oligonucleotides comprising a 5xe2x80x2 primer, a central primer and a plurality 3xe2x80x2 primers that hybridize to different alleles of microsatellite DNAs.
The analysis of variation among polymorphic DNA provides valuable tools for genetic studies in the development of genetic engineering, medicine, gene mapping and drugs. For example, variations in polymorphic DNA allows one to distinguish one individual of a population from another, or to assess the predisposition of an individual to a heritable disease or trait.
Two types of genetic markers widely used in genetic studies include microsatellites and single nucleotide polymorphisms (SNPs). Microsatellites are genomic regions that are distributed approximately every 30 kilobases throughout the genome and that contain a variable number of tandemly repeated sequences of mono, di-, tri-, tetra-, penta-, hexa-, hepta-, octa- or nona-nucleotides. SNPs are found approximately every kilobase in the genome.
SNPs and microsatellites differ in primary DNA structure, relative genome density and genetic information. For example, SNPs are more suitable than microsatellites for genotyping with a high-density of markers because of their distribution and the high sequence specificity possessed by sequences adjacent to the SNP site. Yet, microsatellites are more informative than SNPs because microsatellites typically possess four to sixteen different alleles compared to only two alleles for SNPs.
Presently, the most commonly used methods for genotyping microsatellite markers are gel-based PCR fragment analysis (Shi et al., (1999) Mol. Diagn., 4: 343-351). These methods are relatively more labor-intensive and time-consuming due to gel preparation and gel reading steps, than the method described in this invention. Moreover, the automated DNA genotyping instruments are expensive compared to other forms of detection and do not address the gel reading problems resulting from nucleotide compression. Other methods such as differential hybridization are limited by hybridization to two or more microsatellite markers that share sequences. (see Korkko et al., 1998).
Oligonucleotide Ligation Assays (OLAs) have been used to detect SNPs (Baron et al., 1996, see also U.S. Pat. Nos. 5,242,794 and 5,866,337) or mutations in a gene (Landegren et al., 1988, U.S. Pat. Nos. 4,988,617 and 6,025,139). These OLAs are designed to hybridize contiguously to single-stranded target DNA sequences. Recently, an OLA was developed to genotype microsatellites containing mono- and di-nucleotide repeats (Zirvi et al., 1999a, 1999b, U.S. Pat. No. 6,054,564, WO 98/03673, EP956359).
FIG. 1 demonstrates the methods employed in these previous OLA methods. For example, in the OLA method used for the detection of SNPs, two oligonucleotides are designed to hybridize to the region of the tested site where ligation would occur. The principle in the OLA assay for detection of mutations within a gene is to hybridize multiple short oligonucleotides contiguously throughout the entire gene. An OLA method for genotyping mono- and dinucleotide repeats has been reported where the ligation of the two oligonucleotides was performed at the middle of the repeat. In all of these methods, the presence of a mismatch would prevent hybridization and ligation of the oligonucleotide at or near the location of the mismatch.
The major drawback of using OLA for genotyping microsatellites is that ligation is not a highly discriminating process and background noise can be a significant problem. To circumvent this problem, modified nucleotides (containing nucleoside analogs) near the ligation junction are used to improve the stringency of both the hybridization and the ligation. However, this raises the cost, because relatively long, specific oligonucleotides arc required for these assays. A method that overcomes these disadvantages of OLA would make this approach simpler and more efficient and amenable to the comparative genotyping of pooled DNA samples.
The present invention relates to a method for genotyping different microsatellite DNA markers using ligation of at least three oligonucleotides. In previous inventions, the principle was to hybridize combinations of two, long oligonucleotides contiguously in order to cover the whole sequence of the microsatellite. This requires the costly synthesis of specific, long oligonucleotides for each microsatellite to be genotyped. In addition, the use of modified nucleotides was necessary to achieve specificity. The present invention has eliminated these problems by using combinations of at least three oligonucleotides for each allele at a locus. Collections of limited numbers of oligonucleotides can be used for genotyping many different microsatellites, thus reducing the cost of oligonucleotide synthesis in large genotyping projects. In addition, by using combinations of three oligonucleotides, a high degree of specificity is achieved without the need to use modified nucleoside analogs.
The present invention comprises the steps of providing a sample containing microsatellite DNA; selecting combinations of at least three oligonucleotides that comprise a 5xe2x80x2 primer, a central primer and a 3xe2x80x2 primers; mixing the sample and primers such that the primers and microsatellite DNA hybridize; adding a ligating reagent; and detecting the presence of ligation products that consist of combinations of three oligonucleotides (5xe2x80x2 primer, central primer and 3xe2x80x2 primers) bound together as a single oligonucleotide with a contiguous sequence, reflecting the precise genotype of the microsatellite in the sample, and thus the allele(s) present. In particular, the 5xe2x80x2 primer comprises at least 5 base pairs complementary to the 3xe2x80x2 flanking region of the microsatellite target strand; the central primer is complementary to the repeated region of the microsatellite target strand DNA; and the 3xe2x80x2 primers comprise sequences that are complementary to the 5xe2x80x2 flanking sequence of the microsatellite target strand with the addition, at the 5xe2x80x2 end, varying numbers of repeat units. The nature and the number of repeat units comprising the central primer depend on the nucleotides in the repeated sequence and the number of repeat units of the shortest allele at a given locus. Likewise, the number of repeat units added at the 5xe2x80x2 end of the 3xe2x80x2 primer also depends on the nucleotides in the repeated sequence and the number and identity (length) of the alleles in a population.
The present invention further provides a method for genotyping a DNA pool containing a mixture of different DNA samples of the same microsatellite wherein the microsatellite DNA includes mono-, di-, tri-, tetra- penta-, hexa-, hepta-, octa- or nona-nucleotide repeated alleles. The detection of the allele content of the microsatellite DNA marker within the pooled sample is determined by gel filtration, electrophoresis, mass spectrometry or a gel free analysis.
In one embodiment, the present invention provides a method for genotyping different alleles of a microsatellite DNA wherein the 5xe2x80x2 end of the 5xe2x80x2 primer is labeled with a detectable label. In another embodiment, the method provides detection of different allele of a microsatellite DNA wherein the 5xe2x80x2 or 3xe2x80x2 primer is covalently linked to a functional group, wherein the functional group is capable of specifically binding to a component of a solid support.
The present invention will decrease cost and improve the experimental quality needed to achieve genotyping using high density DNA pooling. The method uses the capacity of DNA ligase to join selectively designed adjacent oligonucleotides that hybridize to a given DNA template. The combination of specifically designed oligonucleotides for each allele within a marker will allow discrimination between the different genotypes.